The contribution of individual domains of chloroplast protein AtDeg2 to its chaperone and proteolytic activities

Przemysław Jagodzik,Robert Luciński,Lucyna Misztal,Grzegorz Jackowski

doi:10.5586/asbp.3570

Przemysław Jagodzik, Robert Luciński + Show 2 more

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https://doi.org/10.5586/asbp.3570

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Abstract

The thylakoid protease AtDeg2 is a non-ATP hydrolyzing chloroplast protease/chaperone peripherally connected with stromal side of thylakoid membrane. Its linear structure consists of protease domain and two PDZ domains. To unveil the significance of individual domains, chaperone and proteolytic activities of AtDeg2, its mutated recombinant versions have been developed and their ability to suppress protein aggregation and resolubilization of protein aggregates as well as the ability to degrade substrate protein was examined in vitro. Our work reveals for the first time that AtDeg2 is able not only to suppress aggregation of denatured proteins, but to resolubilize existing protein aggregates as well. We show that PDZ2 domain contributes significantly to both chaperone and protease activities of AtDeg2, whereas PDZ1 is required for chaperone but superfluous for proteolytic activity. Protease domain – but not S-268 in its catalytic site – contributes to chaperone activities of AtDeg2. These results show an entirely new function of AtDeg2 chaperone/protease (i.e., disaggregation of protein aggregates) and allow to identify structural motifs required for “old” and new functions of AtDeg2.

Highlights

Non-ATP hydrolyzing Deg serine endopeptidases were first discovered and elaborated as a result of studies performed on E. coli mutants which had not been able to grow at high temperatures (>37°C) due to inability to degrade periplasmic proteins damaged under such conditions [1,2] and these endopeptidases were later found to occur in cells of all living organisms [3]
Our results indicate that both PDZ domains and the protease domain excluding catalytically active S-268 are required for AtDeg2 to exhibit chaperone activities and that PDZ1 is unnecessary to maintain proteolytic activity
Our study revealed for the first time that AtDeg2 exhibits a potency to resolubilize already existing lysozyme aggregates as well (Fig. 2). This finding provides significant insight into AtDeg2 function since only one non-ATP hydrolyzing chaperone able to induce protein disaggregation has been identified previously, namely the 43-kDa subunit of a chloroplast cpSRP43/cpSRP54 heterodimer, which was found to be responsible for targeting of precursors of LHCPII to thylakoid membrane [22,23]

Summary

Introduction

Non-ATP hydrolyzing Deg serine endopeptidases were first discovered and elaborated as a result of studies performed on E. coli mutants which had not been able to grow at high temperatures (>37°C) due to inability to degrade periplasmic proteins damaged under such conditions [1,2] and these endopeptidases were later found to occur in cells of all living organisms [3]. Sixteen genes coding for proteins orthologous to E.coli Deg proteases (DegP, DegQ, and DegS) have been identified in the A. thaliana nuclear genome and marked AtDEG1–16 [4]. It has been confirmed both by GFP tagging and by proteomic studies that products of AtDEG 1, 2, 5, 8, and 13 are targeted exclusively to chloroplasts with AtDeg being sorted to stromal side and AtDeg , and 8 to luminal side of thylakoid membrane [5,6,7,8]. As judged by the results of studies based on GFP tagging approach, remaining AtDEG

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